NO donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction, and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation, or if some contribution occurs via cGMP/PKG-independent mechanisms, in indo-1 loaded adult cardiac myocytes. While a high concentration of the NO donor, S-nitroso N-acetyl-penicillamine (SNAP, 100 uM), significantly attenuated contraction amplitude (TA) by 24.4 +/- 4.5% (without changing the Ca2+ transient (CaT) or total cAMP), a low concentration of SNAP (1 umol/L) significantly increased TA (38 +/- 10%), CaT (26 +/- 10%), and cAMP levels (from 6.2 to 8.5 pmol/mg protein). The negative contractile response of 100 umol/L SNAP was completely abolished in the presence of the specific blocker of PKG, KT 5823 (1 umol/L); the positive contractile response of 1 umol/L SNAP persisted despite the presence of the selective inhibitor of GC, ODQ (10 umol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 umol/L). Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1-1 umol/L) of SNAP (AC, 18-20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP at least in part by a novel cGMP-independent activation of AC and induces a positive contractile response.